Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 Research Article OPEN ACCESS Freely available online doi:10.4172/jpb.1000098 Proteomic Alterations During Dormant Period of Longa Rhizomes Daranee Chokchaichamnankit1, Pantipa Subhasitanont1, N. Monique Paricharttanakul1, Polkit Sangvanich2, Jisnuson Svasti1,3,4, Chantragan Srisomsap1,3,* 1Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand 2Research Center for Bioorganic Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 3Chulabhorn Graduate Institute, Bangkok 10210, Thailand 4Department of Biochemistry and Center for Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand firmed by contemporary scientific research. These include an- Abstract tioxidant, antiarthritic, antimutagenic, antitumor, antithrombotic, antivenom, antibacterial, antifungal, antiviral, nematocidal, Proteins involved during storage of Curcuma longa choleretic, antihepatotoxic and hemagglutinating activities have not been investigated in detail. Proteins and nucleic (Kumar et al., 2006; Sangvanich et al., 2007). acids are considered to be essential for the sprouting period. As numerous applications of proteomic ap- In physiology, dormancy is a period of arrested plant proaches have been reported in many areas of biology, growth. It is a strategy exhibited by many plant species to en- biochemistry and biomedicine, we chose to use able their survival in climates unsuitable for growth such as proteomic technology to study the protein expression winter or dry season. will start going dormant (rest- of Curcuma longa (Khaminchun) rhizome during dor- ing) in the fall (October), even in warm tropical climates and mancy and sprouting. Microscale solution-phase iso- the rhizomes will grow during summer (Panneerselvam, 1998). The levels of sugars, starch and the activities of enzymes in- electric focusing (Zoom) was employed to enrich the volved in carbohydrate metabolism, respiration, glycolysis, low abundance proteins in the pH range of 5.4-10 and tricarboxylic acid (TCA) cycle and oxidative pentose phosphate improve the separation of those proteins in the acidic pathway (PPP) have been studied during the early period of range from 3-5.4. Samples were drawn at seven-day dormancy and sprouting (Panneerselvam et al., 2007). intervals from harvest until the commencement of sprouting. The proteomic patterns of the storage period At the molecular level, little is known regarding the protein (0, 14, 21, 42, and 70 days) were studied and identified changes during storage of Curcuma longa. Proteins and nucleic by LC/MS/MS in these two pH ranges. High levels of acids are considered to be essential for the sprouting of potato buds (Tuan and Bonner, 1964). As numerous applications of glyceraldehyde-3-phosphate dehydrogenase, a glyco- proteomic approaches have been reported in many areas of bi- lytic enzyme, were present and in glycosylated and phos- ology, biochemistry and biomedicine, we chose to use phorylated forms. Sporamin, the major storage protein proteomics to study the protein expression of Curcuma longa of the tuberous roots of sweet potato, was highly ex- (Khaminchun) rhizome during dormancy and sprouting. pressed in the dormant period and lower expression was Microscale solution-phase isoelectric focusing (Zoom) was em- detected in the sprouting period. Moreover, the enzyme ployed to enrich the low abundant proteins in the pH range for catalyzing the synthesis of catechin, from 5.4-10 and separate the acidic group (3-5.4) of proteins as leucoanthocyanidin reductase, was found for the first well. The two pH ranges of proteins of different periods of dor- time in the Curcuma longa rhizome. These results rep- mancy of Curcuma longa were compared and proteins identi- resent the first proteomic patterns during the storage fied using LC/MS/MS. period of Curcuma longa. For the rhizomes of Curcuma *Corresponding author: Dr. Chantragan Srisomsap, Laboratory of Bio- longa, visible sprouting was observed within 70 days chemistry, Chulabhorn Research Institute, Vibhavadee Rangsit Road, after harvest. Bangkok 10210, Thailand, Tel: (66-2)-5740622 to 33 ext. 3715; Fax: (66-2)-5740625 ext. 3716, E-mail: [email protected] Keywords: ; Curcuma; Rhizomes; Enrich; Dor- Received July 10, 2009; Accepted September 23, 2009; Published mancy; Sprouting September 25, 2009 Citation: Chokchaichamnankit D, Subhasitanont P, Paricharttanakul NM, Introduction Sangvanich P, Svasti J, et al. (2009) Proteomic Alteration During Dor- mant Period of Curcuma Longa Rhizomes. J Proteomics Bioinform 2: Curcuma is a perennial herb of the family Zingiberaceae. The 380-387. doi:10.4172/jpb.1000098 rhizome of Curcuma, commonly called (Curcuma Copyright: © 2009 Chokchaichamnankit D, et al. This is an open-ac- longa L.), has been used for centuries as spice and coloring cess article distributed under the terms of the Creative Commons Attri- agent. Turmeric is indigenous to South, Southwest and South- bution License, which permits unrestricted use, distribution, and repro- east Asia. The traditional uses of turmeric in folk medicines are duction in any medium, provided the original author and source are cred- multiple and many of the therapeutic effects have been con- ited. J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 380 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009

Methods Two-dimensional Polyacrylamide Gel electrophoresis (2-D PAGE) Protein Extraction 2-D PAGE was performed using the immobiline/polyacryla- Fresh rhizomes of Curcuma longa were harvested from mide system. Samples were applied by overnight in-gel Ratchaburi province after the aerial parts have fully dried up rehydration of 70 mm ( analytical runs ). pH 4-7 IPG gel strips and stored in the dark. This period is considered to be the initial (GE Healthcare, Biosciences, Uppsala, Sweden) were used for period of dormancy. Mature healthy rhizomes of uniform size samples not enriched by Zoom, whereas non-linear pH 3-10 and weight were kept at 28±2ºC and relative humidity of 65- and 3.9-5.1IPG gel strips were used for samples enriched. The first dimen- 75% in a biophotochamber. Samples were collected at 7-day sion (IEF) was performed at 6,500 Vh, using a Pharmacia LKB intervals from harvest until sprouting. Multiphor II system. The IPG strips were equilibrated in two steps of equilibration buffer. The first step employed 50 mM Curcuma longa rhizomes (15 grams) were peeled to remove Tris-HCl buffer, pH 6.8, 6 M urea, 30% glycerol, 1% SDS and the skin before ground in liquid nitrogen using a mortar and 1% DTT, while 2.5% iodoacetamide replaced DTT in the pestle, and suspended in 30 ml of extraction buffer (0.7 M su- second step. The IPG strips were then applied to the crose, 0.5 M Tris-HCl, 30 mM HCl, 0.1 M KCl and 1% v/v β- second dimension 14% T SDS polyacrylamide gels mercaptoethanol). The mixture was extensively homogenized (100x80x1.5mm). Electrophoresis of the mini-gel was per- and stored at 4°C for at least 30 minutes. After centrifugation at formed in a Hoefer system at 20 mA for two hours at room 4,000g for 10 minutes, the supernatant was transferred and kept temperature. After electrophoresis, proteins were visualized by at 4°C. The plant tissues were resuspended in extraction buffer CBR-250 staining. two times. Fifty milliliters of water-saturated phenol was added to the supernatant, mixed and kept at 4°C for 60 minutes. After Gel Scanning and Image Analysis centrifugation at 8,000g for 10 minutes, the upper phenol phase was removed and resuspended twice in water-saturated phenol Gels were scanned using an ImageScanner II (GE Healthcare, (Hurkman and Tanaka, 1986). Proteins were precipitated from Uppsala, Sweden). The ImageMasterTM (GE Healthcare, the phenol phase of 200 ml 0.1 M ammonium acetate, dissolved Uppsala, Sweden) was used for computer analysis. in methanol and stored overnight at – 20°C. The sample was Tryptic In-gel Digestion centrifuged at 4,000g for 10 minutes. The pellet was dissolved in 10 ml of cold water and sonicated for 3 minutes. The pro- Protein spots were excised and transferred to 0.5 ml microfuge teins were precipitated in 9 volumes of cold acetone. The solu- tubes. Fifty µl of 0.1 M NH4HCO3 in 50% acetonitrile was tion was left at -20 C for at least 4 hours and then centrifuged added. The gel was incubated 3 times for 20 minutes at 30°C. at 4,000g for 10 minutes. The supernatant was discarded and The solvent was discarded and gel particles were dried com- the pellet was air-dried briefly until the smell of acetone had pletely by Speed Vac. Reduction and alkylation was performed disappeared. The amount of protein was determined by using by swelling the gel pieces in 50 µl buffer solution (0.1 M Bradford method. NH4HCO3, 10 mM DTT and 1 mM EDTA) and incubating at 60°C for 45 minutes. After cooling, the excess liquid was re- Microscale Solution-phase Isoelectric Focusing (Zoom) of moved and quickly replaced by the same volume of freshly Protein Extract prepared 100 mM iodoacetamide in 0.1 M NH4HCO3 solution. For in-solution IEF fractionation, a microscale solution-phase The reaction was incubated at room temperature in the dark for isoelectric focusing (ZOOM) IEF fractionator was used 30 minutes. The iodoacetamide solution was removed and the (Invitrogen, Carlsbad, CA, USA). Proteins were reduced by gel pieces were washed with 50% acetonitrile in water, 3 times dissolving with 50 mM DTT (USB Corporation Cleveland, OH, for 10 minutes each time, and the gel pieces were completely USA), 7.7 M urea (ICN), 2.2 M thiourea (Sigma, St Louis, MO, dried. Aliquots of trypsin (Promega Corporation, WI, USA) (1 USA), and 4.4% CHAPS (Calbiochem, San Diego, CA, USA). µg trypsin /10 µl 1% acetic acid) were prepared and stored at The reduced proteins were then alkylated by adding 100 mM -20°C Fifty µl of digestion buffer (0.05 M Tris HCl, 10% ac- iodoacetamide for 30 min and incubated in the dark. Insoluble etonitrile, 1 mM CaCl2, pH 8.5) and 1 µl of trypsin were added matter was removed by 20 min centrifugation at 12,000 rpm. to the gel pieces. After incubating the reaction mixture at 37°C To remove iodoacetamide, the protein lysate was precipitated overnight, the digestion buffer was removed and saved. The using cold acetone. The pellet was resuspended in 7.7 M urea, peptides were then extracted from the gel pieces by adding 60 2.2 M thiourea, 4.4% CHAPS, 10 mM DTT and 0.8% ampholine µl of 2% freshly prepared trifluoroacetic acid and incubating at an approximate protein concentration of 0.6 mg/ml. The for 30 minutes at 60°C. The extract and the saved digestion Zoom-IEF fractionator was assembled with disks pH 3.0, pH buffer were finally pooled and dried. 5.4 and pH 10.0. One thousand and three hundred µl lysate was Protein Identification by LC/MS/MS loaded between disk pH 3.0-5.4 and 650 µl was loaded be- tween disk pH 5.4-10.0. The proteins were focused for 20 min LC/MS/MS analyses were carried out using a capillary LC at 70 V, 160 min at 70-600 V, and 120 min at 600 V. After system (Waters) coupled to a Q-TOF mass spectrometer separation each fraction was transferred into a clean (Micromass, Manchester, UK) equipped with a Z-spray ion- microcentifuge tube. source working in the nanoelectrospray mode. Glu-fibrinopep- tide was used to calibrate the instrument in MS/MS mode. The

J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 381 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 tryptic peptides were concentrated and desalted on a 75 µm ID x 150 mm C18 PepMap column (LC Packings, Amsterdam, 4 Isoelectric point 7 kDa The Netherlands). Eluents A and B were 0.1% formic acid in 3% acetonitrile and 0.1% formic acid in 97% acetonitrile, re- 97 spectively. Six µl of sample was injected into the nanoLC sys- 66 tem, and separation was performed using the following gradi- 45 ent: 0 min 7% B, 35 minutes 50% B, 45 minutes 80% B, 49 minutes 80% B, 50 minutes 7% B, 60 minutes 7% B. The data- base search was performed with ProteinLynx screening SWISS- 30 PROT and NCBI. For some proteins that were difficult to find, the Mascot search tool available on the Matrix Science site screening NCBInr was used. 20 Glycoprotein Gel Staining

After two-dimensional electrophoresis, the gel was immersed 14 in fixing solution (50% methanol and 5% acetic acid) with gentle agitation at room temperature for 45 minutes. This fixation step was repeated overnight and washed twice with 3% glacial ace- tic acid at room temperature for 20 minutes. The gel was then incubated in oxidizing solution (periodic acid in 3% acetic acid) Figure 1: Representative two-dimensional gel electrophore- at room temperature for 30 minutes and then washed twice with sis analysis of proteins isolated from the rhizomes of Curcuma 3% glacial acetic acid at room temperature for 20 minutes. The longa at pH range 4-7. gel was stained with freshly prepared Pro-Q Emerald 300 Stain- ing Solution (Invitrogen, Carlsbad, CA, USA) in the dark for 2 and Pelpoir, 2007). Therefore, the phenol extraction method hours at room temperature. Finally, the gel was washed again was selected for prior protein preparation for all experiments. twice before visualizing using Ettan DIGE Imager (GE Healthcare). Enrichment of Low Abundant Proteins Using Microscale Solution-phase Isoelectric Focusing (Zoom) Phosphoprotein Gel Staining Initially, total proteins from the rhizomes of Curcuma After two-dimensional electrophoresis, the gel was fixed, were run on 2-DE using pH 4-7 linear strips. However, the washed with ultrapure water and incubated in Pro-Q Diamond proteomic patterns show high intensity spots in the acidic re- phosphoprotein gel stain (Invitrogen, Carlsbad, CA, USA) in gion and weak spots in the weakly acidic to basic region (Fig- the dark for 90 minutes. The gel was destained with 20% ac- ure 1). Therefore, enrichment of the low abundant proteins in etonitrile, 50 mM sodium acetate, pH 4 for 30 minutes at room the pH range 5.4-10 was pursued using microscale solution- temperature, protected from light. After washing with ultrapure phase isoelectric focusing (Zoom). water, the stained gel was visualized on a Typhoon Imager (GE Healthcare). Protein Expression and Identification During the Storage Period (0-70 days) of Curcuma Longa Results and Discussion Rhizomes of Curcuma longa were collected from the day of This study was aimed to investigate the changes in proteins harvest (day 0) until the commencement of sprouting from the during the dormant period of Curcuma longa as the role of buds (day 70). Protein expression during the storage period (0- proteins regulating its dormancy is unclear. We employed 70 days) of Curcuma longa was analyzed by two dimensional proteomic technology to study the protein expression from the gel electrophoresis (2-DE) at day 0, day 14, day 21, day 49 and day of harvest (day 0) to the commencement of sprouting (day day 70. Figure 2(included as a supplementary information) 70) of Curcuma longa. The total storage period was 70 days. shows the 2-DE patterns in the pH range 3-5.4 and Figure 3(in- Sample Preparation for 2-D PAGE cluded as a supplementary information) represents the patterns in the pH range 5.4-10 for each of the days analyzed. Most of Proteomic analysis of plant samples still has some limita- the dark spots from the range of pH 3.0-5.4 were identified by tions especially with regards to sample preparation. The inter- LC/MS/MS but some spots did not match with any protein in ference of compounds such as tannins, polyphenols, lignins, database and could not be identified. Twenty-four spots in the alkaloids and pigments can prevent obtaining good resolution range of pH 3.0-5.4 and 42 spots in the range of pH 5.4-10 in 2-DE gels. Three solvent systems, namely acid-acetone, phe- patterns of Curcuma longa were reported in Table 1(included nol and trichloroacetic acid, were evaluated for precipitating as a supplementary information). The spot numbers represent proteins from the rhizomes of Curcuma plants (data not shown). the spots cut from gels and identified by using LC/MS/MS. Among these solvent systems, phenol extraction is the most The identified proteins were then grouped with regards to suitable solvent as it allows efficient protein recovery and re- their functional classification based on the following catego- moves non-protein components in the case of plant tissues rich ries: metabolism, photosynthesis, stress response, cell struc- in polysaccharides, lipids, and phenolic compounds (Faurobert J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 382 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 ture, protein synthesis, defense, transport and unannotated/func- nase small subunit (spot no. 2), a protein involved in the Calvin tion inferred. Unannotated/function inferred include a number cycle or the 3-carbon pathway, was found. There were two pro- of peptides, which have not been ascribed functions in data- teins involved in the 4-carbon pathway, i.e., NADP-dependent bases. Relevant information on the functions of proteins was malic enzyme (spot no. 28) and phosphoenolpyruvate carboxy- taken from SWISS-PROT and NCBI. Figure 4 represents the lase (spot no. 53). functional distribution of the identified proteins expressed in Curcuma longa rhizomes. Among these functional classes, Sporamin A precursor (spot no. 13), the major storage pro- 43.9% were metabolic proteins whereas proteins involved in tein of the tuberous roots of sweet potato, was present. Proteins protein synthesis, defense, stress response, photosynthesis, involved in isoprenoid biosynthesis pathway, such as putative transport and cell structure were found to be 9.1%, 7.6%, 6.1%, sesquiterpene cyclase (spot no. 9) and farnesyl pyrophosphate 7.6%, 1.5% and 1.5%, respectively. The remaining 22.7% were synthase 2 (spot no. 31), were found in the rhizomes. The ex- annotated as unknown. pression of the enzyme for catalyzing the synthesis of catechin, leucanthocyanidin reductase (spot no. 58), was found for the In the dormant period, less than half of all proteins identified first time in the rhizomes. Proteins involved in stress response are involved in metabolism namely, glycolysis, gluconeogen- were 18.3, 18.1 and 17.6 kDa class I heat shock proteins (spot no. 39, 40, 41).

Cell structure 1.52% Differential Protein Expression During Dormancy and Defense Sprouting of Curcuma Longa 7.58% Stress response Unknown 6.06% 22.73% Using ImageMasterTM computer analysis, the percent volumes Transport of each spot at day 0, 14, 21, 49 and 70 days were determined 1.52% and shown in Figure 5. The enzymes involved in carbohydrate metabolism were found in the rhizomes of Curcuma longa.

Protein synthesis Enzymes involved in glycolysis were present from day 0, e.g., 9.09% glyceraldehyde-3-phosphate dehydrogenase (GADPH), phos- Metabolism 43.94% phoglycerate kinase, triose phosphate isomerase and enolase, Photosynthesis and all of these enzymes were present at day 70 when sprout- 7.58% ing was observed. The highest level of all proteins detected Figure 4: Functional distribution of identified proteins ex- was GAPDH (spot no. 25, 45), a classical glycolytic enzyme, involved in cellular energy production and has important house- pressed in Curcuma longa rhizomes. keeping function. The expression levels of GAPDH were maxi- mal at day 14 and decreased upon sprouting. Another spot (spot esis and starch degradation. Multiple spots were detected for no. 46) was detected at lower levels. The level of another gly- proteins involved in glycolysis, namely, glyceraldehyde-3-phos- colytic enzyme, fructose biphosphate aldolase (spot no. 26), phate dehydrogenase (3 forms, spot no. 25, 45, 46), fructose was also highest at day 14 and gradually decreased. biphosphate aldolase (spot no. 26), enolase (2-phosphoglycer- ate hydratase) (3 forms, spot no. 29, 54, 55), phosphoglycerate Enzymes involved in gluconeogenesis and starch degrada- kinase (3 forms, spot no. 32, 47, 56) and triose phosphate tion pathway were also detected in our analysis at day 70. This isomerase (spot no. 36). Two proteins involved in gluconeo- result correlates well with a study performed in which the starch genesis were detected, i.e., alcohol dehydrogenase II (spot no. content in the tubers of D. esculenta decreased as sprouting 37) and alcohol dehydrogenase (spot no. 66). Two proteins in- initiated. It showed that when a single sprout grows, it draws volved in starch degradation were isoamylase (spot no. 38) and upon the carbohydrates in every part of the tuber simultaneously alpha-glucan phosphorylase, H isozyme (spot no. 52). Three (Davis and Ross, 1984). A comparative analysis of starch and proteins involved in amino acid biosynthesis were s- sugars during the dormant period until sprouting indicates that adenosylhomocysteine hydrolase (spot no. 48), threonine syn- the total sugars present increased steadily as sprouting pro- thase chloroplast precursor TS (spot no. 50) and s- gressed while the starch content decreased. A similar trend was methyltetrahydropteroyltriglutamate-homocysteine also reported in potato tubers (Copp et al., 2000). Accumula- methyltransferase (spot no. 51). tion of sugars was observed in Lilium rubellum bulbs during dormancy and it increased to higher levels during sprouting Six proteins responsible for protein synthesis with major roles (Xu et al., 2006). The carbohydrates and sugar contents of Cur- in controlling cell growth division and development were iden- cuma alismatifolia Gagnep were studied during their dormant tified, which include 60S ribosomal protein L10 (spot no. 33), period (Ruamrungsri et al., 2001). The hydrolysis of starch in ribosomal protein subunit 2 (spot no. 34), ribosomal protein S7 the rhizome and storage roots in the middle of the dormant (spot no. 44), ribosomal protein S18 (spot no. 65), maturase K period was observed, however total soluble sugars did not in- (spot no. 10) and beta subunit of RNA polymerase (spot no. crease. It was therefore suggested that active utilization of car- 23). There were four proteins involved in photosynthesis, bohydrates was essential for shoot growth in both rhizome and namely, photosystem I assembly protein (spot no. 5), putative storage roots during dormancy. PSII-P protein (spot no. 20), oxygen-evolving enhancer protein 2 (spot no. 4) and putative oxygen-evolving enhancer protein 1 Proteins involved in amino acid biosynthesis, namely, S- (spot no. 18). Ribulose 1, 5-bisphosphate carboxylase/oxyge- adenosyl-Lhomocysteine (SAH) hydrolase (spot no. 48), threo-

J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 383 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 turn is hydrolyzed to L-homocysteine and adenosine. High SAH 18 A. Glycolysis 16 hydrolase activities in cell-free extracts from young tea leaves 14 0d 12 were detected. 14 d

Vol 10 21 d

% 8 49 d Maturase K (spot no. 10) and the beta subunit of RNA poly- 6 70 d 4 merase (spot no. 23) were detected as well in relatively high 2 amounts during storage, but the levels started to decrease upon 0 25 26 29 32 36 45 46 47 54 55 56 sprouting (day 70). These proteins are significant for control- Spot number ling protein synthesis along with the other ribosomal proteins detected. The photosynthetic proteins were found from day 0, 6 B. Gluconeo- C. Starch D. Amino acid genesis degradation biosynthesis i.e., photosystem I assembly protein ycf4 (spot no. 5), plasto- 5 0d cyanin-like (spot no. 16), oxygen-evolving enhancer protein 1 4 14 d and 2 (spot no. 18 and 4). Only one protein involved in the Vol 3 21 d % 49 d Calvin cycle or the C-3 pathway, i.e., ribulose 1, 5-bisphosphate 2 70 d carboxylase/oxygenase small subunit (spot no. 2) was detected, 1 and the levels increased from day 0 to day 70. Differential ex- 0 37 66 38 52 48 50 51 pression was observed for proteins involved in C-4 pathway. Spot number NADP-dependent malic acid (spot no. 28) was found from day 0 to day 70 but phosphoenolpyruvate carboxylase (spot no. 53) 8 E. Protein synthesis was present at day 70. Usually plants in the tropical areas such 7 6 0d as sugar cane, rice and maize are called C4 grasses. The mo- 5 14 d lecular basis for C4 differentiation is still poorly understood. Vol 4 21 d % 3 49 d Differential protein accumulation and activities between meso- 2 70 d phyll and the bundle sheath chloroplast of maize and sorghum 1 (both NADP-Malic enzyme (ME) type C4) have been studied 0 10 23 33 34 44 65 using various low throughput techniques (Hatch and Slack, Spot number 1966).

G. 3-Carbon H. 4-Carbon 6 F. Photosynthesis The proteins involved in isoprenoid biosynthesis such as pu- pathway pathway tative sesquiterpene cyclase (spot no. 9), farnesyl pyrophosphate 0d 4 14 d synthase 2 (spot no. 31), were present in the rhizome during the

Vol 21 d whole storage period. The content of bisabolane-type sesquit- % 2 49 d erpenes in volatile oil was found in high amount for Curcuma 70 d longa (Zeng et al., 2007). The key step in terpenoid biosynthe- 0 sis is the conversion of acyclic prenyldiphosphates to terpenoid 5 16 20 4 18 2 28 53 Spot number compounds by specific terpenoid synthase (cyclases) (Shen et al., 2000). Sesquiterpene cyclase, a branch point enzyme in the J. Isoprenoid K. Catechin 10 I. Storage L. Stress response general isoprenoid pathway for the synthesis of phytoalexin biosynthesis synthesis capsidiol, was induced in detached leaves of Capsicum annuum 8 pathway 0d (pepper) by UV treatment. The inducibility of cyclase enzyme 6 14 d Vol 21 d activities paralleled the absolute amount of cyclase protein(s) % 4 49 d of pepper immunodetected by monoclonal antibodies raised 2 70 d against tobacco sesquiterpene cyclase (Back et al., 1998). 0 13 9 31 58 39 40 41 Leucoanthocyanidin reductase (spot no. 58), the NmrA-type Spot number oxidoreductase family or isoflavone reductase subfamily was Figure 5: The quantifications of the proteins involved in glyco- first found by our group in the rhizome of sprouting period of lysis (A), gluconeogenesis (B), starch degradation (C), amino Curcuma longa. This protein catalyzes the synthesis of catechin acid biosynthesis (D), protein synthesis (E), photosynthesis (F), from 3,4-cis-leucocyanidin. Cathechins are polyphenolic anti- 3-carbon pathway or calvin cycle (G), 4-carbon pathway (H), oxidant plant metabolites. These compounds are abundant in storage protein (I), isoprenoid biosynthesis (J), catechin synthe- teas derived from the tea-plant Camellia sinensis as well as in sis (K) and stress response (L). some cocoas and chocolates made from the seeds of Theobroma cacao (Punyasiri et al., 2004). nine synthase chloroplast precursor TS (spot no. 50) and S- Heat shock proteins were found as sHsp family with molecu- methyltetrahydropterpyl-triglutamate-homocysteine lar weight 18.3, 18.1 and 17.6 kDa (spot no. 39, 40, 41). In methyltransferase (spot no. 51), were prominent during dor- plants, sHsps form a more diverse family than other Hsps/chap- mancy and early sprouting. The new caffeine biosynthetic path- erones with respect to sequence similarity, cellular location and way in tea leaves was reported on the utilization of adenosine functions (Wang et al., 2004; Waters et al., 1996; Vierling, 1991). released from the S-adenosyl-L-methionine (SAM) cycle sHsp functions by preventing aggregation and stabilizing non- (Koshiishi et al., 2001). SAM is converted to SAH, which in native proteins. Under normal growth conditions, most sHsps J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 384 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 cannot be detected in the vegetative tissues, but are rapidly pro- The presence of post-translational modifications in proteins duced in response to heat. Increasing the temperature to approxi- in the 70-day Curcuma longa rhizomes can be detected by gly- mately 10-15ºC above the optimal growth temperature, which coprotein (Figure 6A, C) and phosphoprotein (Figure 6B, D) is usually in the sub-lethal range, induces the heat shock re- staining of the 2-DE gels of proteins enriched at the acidic and sponse. basic ranges, respectively. Some proteins enriched at pH 3.0- 5.4 that were positive for glycosylation included ribulose car- Vegetative storage organs such as bulbs, tubers, corms, rhi- boxylase/oxygenase small subunit (spot no. 2), photosystem I zomes and bark act as sinks for soluble nitrogen compounds assembly protein ycf4 (spot no. 5), peptidyl-prolyl cis trans (mainly amino acids) generated from the leaf proteins when the isomerase-like (spot no. 6), actin (spot no. 8), putative sesquit- plant enters a senescing phase (Van Damme et al., 2000). These erpene cyclase (spot no. 9), F-box protein interaction domain storage organs become a source of nitrogen when the plant re- (spot no. 12), sporamin A precursor (spot no. 13), peptidase M sumes growth after a resting or dormancy period. There are re- (spot no. 15) and beta subunit of RNA polymerase (spot no. 23) ports of increased 1-2% dry weight in cassava and up to 10% as shown in Figure 6A. Most of those spots were also positive dry weight in yam bean (Shewry, 2003). Classical examples of for phosphorylation except for actin (spot no. 8), putative ses- storage proteins are the tuber storage proteins from potato quiterpene cyclase (spot no. 9) and beta subunit of RNA poly- (Solanum tuberosum) and sweet potato (Ipomoea batatas), which merase (spot no. 23), with the presence of phosphorylation of are commonly named as patatin and sporamin, respectively plastocyanin-like protein (spot no. 16) as shown in Figure 6B. (Mignery et al., 1984; Maeshima et al., 1985). From our result, sporamin, the major storage protein of the tuberous roots of sweet For enriched pH 5.4-10 proteins shown in Figure 6C, six pro- potato, was highly expressed during the storage period in teins were found to be positive with glycoprotein staining, which Curcuma longa, with the highest level at day 49. Sporamin was are ribosomal protein subunit E (spot no. 34), vacuolar ATP reported to have antioxidant activity, acting as a synthase (spot no. 35), alcohol dehydrogenase II (spot no. 37), dehydroascorbate reductase and monodehydroascorbate reduc- isoamylase (spot no. 38), glyceraldehyde-3-phosphate-dehydro- tase, and that this was associated with intermolecular thiol/dis- genase (spot no. 45, 46), and 5-Methyltetrahy- ulfide exchange (Hou and Lin, 1997). dropteroytriglutamatehomocysteine methyltransferase (spot no. 51). Seven proteins in Figure 6D, glyceraldehyde-3-phosphate- Post-translational Modifications of Proteins During Storage dehydrogenase (spot no. 25, 45), fructose biphosphate aldolase Period (0-70 days) of Curcuma Longa

3.9 5.1 3.9 5.1 pI pI k Da 97 66

45

30

20

14

3 pI 10 3 pI 10 k Da

97 66

45

30

20

14

Figure 6: Identification of post-translational modifications present in Curcuma longa rhizomes. The 2-DE gels of the enriched pH 3.0-5.4 proteins and the enriched 5.4-10 proteins from 70 days of the Curcuma longa rhizomes were stained with Pro-Q Emerald 300 Staining Solution for glycoprotein detection (A, C) and Pro-Q Diamond phosphoprotein gel stain (B, D), respectively. The identity of each labeled spot is listed in Table 1.

J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 385 ISSN:0974-276X JPB, an open access journal Journal of Proteomics & Bioinformatics - Open Access JPB/Vol.2/September 2009 (spot no. 26), formate dehydrogenase 1 (spot no. 27), 18.3 kDa Electrophoresis. Plant Physiol 81: 802-806.» CrossRef » Pubmed class I heat shock protein (spot no. 39), Sadenosylhomocysteine » Google Scholar hydrolase (spot no. 48), threonine synthase chloroplast precur- 8. Koshiishi C, Kato A, Yama S, Crozier A, Ashihara H (2001) sor TS (spot no. 50) and leucoanthocyanidin reductase (spot no. A new caffeine biosynthetic pathway in tea leaves: utilisation 58) were found to be positive with phosphoprotein staining. of adenosine released from the S-adenosyl-L-methionine cycle. FEBS Lett 499: 50-54. » CrossRef » Pubmed » Google Scholar Conclusions 9. Kumar GS, Nayaka H, Dharmesh SM, Salimath PV (2006) For the rhizomes of Curcuma longa, visible sprouting was Free and bound phenolic antioxidants in amla (Emblica observed within 70 days after harvest. The first proteomic pat- officinalis) and turmeric (Curcuma longa). J Food Com Anal terns during the dormancy period until sprouting (day 70) of 19: 446-452.» CrossRef » Google Scholar this plant were studied. Two forms of glyceraldehyde-3-phos- phate dehydrogenase were present at high levels as isotype and 10. Maeshima M, Sasaki T, Asahi T (1985) Characterization of only one form showed both glycosylated and phosphorylated pat- major proteins in sweet potato tuberous roots. Phytochemis- tern. Sporamin, the major storage protein of the tuberous roots try 24: 1899-1902.» CrossRef » Google Scholar of sweet potato was highly expressed in the dormant period and lower expression seen in the sprouting period. The expression 11. Mignery GA, Pikaard CS, Hannapel DJ, Park WD (1984) of putative sesquiterpene cyclase and farnesyl pyrophosphate Isolation and sequence analysis of cDNAs for the major synthase 2, involved in the isoprenoid biosynthesis, were found in the potato tuber protein, patatin. Nucleic Acids Res 12: 7987- dormant period. The enzyme for catalyzing the synthesis of cat- 8000.» CrossRef » Pubmed » Google Scholar echin, leucoanthocyanidin reductase, was found for the first time in the Curcuma longa rhizome. Further investigation of other 12. Panneerselvam R (1998) Physiology of seed and bud dor- species will be useful to determine species-specific questions. mancy in: A. Hemantaranajan (Ed.) Advances in Plant Physi- ology, Scientific Publishers, India, pp.419-438. Acknowledgements 13. Panneerselvam R, Jaleel CA, Somasundaram R, Sridharan This investigation was supported by the Chulabhorn Research R, Gomathinayagam M (2007) Carbohydrate metabolism in Institute. We would like to thank Dr. K. Lerdprapamongkol and Dioscorea esculenta (Lour.) Burk. tubers and Curcuma longa Dr. James R. Ketudat-Cairns for helpful discussion. L. rhizomes during two phases of dormancy. Colloids Surf References B Biointerfaces 59: 59-66. » CrossRef » Pubmed » Google Scholar

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J Proteomics Bioinform Volume 2(9) : 380-387 (2009) - 387 ISSN:0974-276X JPB, an open access journal